Extract with freshly equilibrated phenol/chloroform. Spin 8,000 rpm SS-34. Repeat extraction 3-4 times until you see nothing at the interface.

Do 1-2 chloroform extraction.

Precipitate DNA with NaAc/EtOH. Rock the tube gently side to side to have DNA tangle form. If DNA is more than 100 µg you should see white precipitate. If less than 100 g freeze at -70 °C for 10 min. spin down the precipitate.

Wash DNA with 2 ml of 70% EtOH.

Air dry DNA ~20 min in the hood (make sure you don’t over dry it).

Resuspend in ~200 μl TE. May increase the vol. depending on the recovery of DNA.

Carefully determine the concentration of gDNA. The solution is very sticky. I typically take 8-10 µl in 800 µl of TE to determine the concentration.

Digest DNA with 2nd restriction enzyme. Typically 100-200 µl digest with 5-10 fold excess of enzyme for overnight. Add 10 µg tRNA and purify the digested DNA by one extraction with freshly equilibrated phenol/chloroform followed by one chloroform extraction. Air dry and resuspend in 30 µl TE buffer.

Run gel with 15 µg of genomic DNA/lane. 10 µg is often ok, but try to use more.

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